Biofilm: An Alarming Niche in Dairy Industry

Biofilm is an aggregation of microbial cells interconnected by extracellular polymeric substances which accelerate growth on different material surfaces adversely affect the dairy industry. This polymicrobial community contains altered phenotype which differs them from planktonic microbes physiologically. It affects the quality and safety of raw materials and their products. Thus, producing serious problems directly affecting human health. Colonisation at the surfaces of open and closed piping systems, floors, waste, walls, ceilings of the production halls play a major role as barrier in the selection of effective sanitation agents for their control. As a result, the sector is forced to face a havoc economical loss. In fact, the condition is aggravating day-by-day. As microbes growing in a biofilm are highly resistant to antimicrobial agents and host’s immune system, it is obligatory to employ effective methods to retard the biofilm formation. Therefore, immediate appropriate precautionary measures should be adopted to combat the condition and prevent further complications.

The existence of biofilm has been explored for several years in the food industry. The first documentation was done roughly 75 years back in 1943 (Zobell, 1943). Biofilms are sessile microbial communities where microbes live together in association with each other on biotic or abiotic substrates which are bounded by extracellular polysaccharides, proteins, lipids and DNA (Sim˜oes et al., 2010). In other words, simply, bioﬁlms represent an important mode of bacterial life colonizing most of the surfaces in nature. Wet solid surfaces promote biofilm production as the biotic and/or abiotic materials present in the liquid settle over the solid counterpart and attract active microbes to form biofilms (Mu et al., 2014). Wet exteriors are generally covered with extracellular matrix which further aid the attachment of microbes (Bardiau et al., 2016). This matrix enhances microbial survival and even protects them from antimicrobial agents (Chen et al., 2007). Thus, biofilms act as a crucial defensive biota where microbes possess a secure live, multiply invariably and able to yield thermal-resistant enzymes and spores (Felipe et al., 2017).

Characteristics of Biofilm

Biofilms are complex, dynamic and remarkably heterogeneous structures. Different biofilms exhibit different chemical and electrical properties. Moreover the genetic expression is also different in biofilm bacteria as compared to the planktonic bacteria (Costerton et al., 1999; Fox et al., 2005). The cells are able to coordinate among each other via intercellular communication using biochemical signalling molecules (Flemming et al., 2010). Besides, these are generally impervious to nutritional and oxidative stresses, desiccation, UV light exposure and sanitizing agents (Fatemi and Frank, 1999). Stainless steel, polyvinyl chloride, polyurethane are the prolific surfaces for biofilm attachment when they come in contact with food materials (Mustapha and Liewen, 1989; Midelet and Carpentier, 2002). The biofilm associated microbes are also much less susceptible to antimicrobial agents than those present in planktonic state. As a result, it becomes difficult to get rid of biofilms from the contact surfaces of food (Simões et al., 2006).

Biofilms endorse intraspecies and/or interspecies interaction and some of them may be formed by aggregation of many bacterial genuses and are known as mixed biofilms (Sim˜oes et al., 2010). A wide variety of foodborne pathogens are also able to attach, colonize and form biofilms, such as the O157 and non-O157 Shiga toxin–producing E. coli (STEC), S. enterica, L. monocytogenes, etc. (Wang, 2019).

Vulnerable Zones of Biofilms Formation

Almost all branches of food industry are affected with it. But it is of utmost concern especially in food processing plants and considerably in dairy industry (Frank et al., 2003; Jessen and Lammert, 2003). In dairy plant, biofilms can develop on floors, walls, drains, interior and exterior of dairy equipment and machines, transporting pipes and even working surfaces are prone to biofilm formation (Krysinski et al., 1992; Hood and Zottola, 1997; Gibson et al., 1999; Sinde and Carballo, 2000; Verran, 2002). These can be seen in almost all product contact surfaces like milk cups at dairy farms to heat exchangers at the processing plants. This poses a great risk to safety, security and quality of dairy products and to the health of consumers who get exposed to such products (Flint et al., 1997; Scheldeman et al., 2005; Burgess et al., 2010., Bayoumi et al., 2012).

Process of Biofilm Formation

The formation process of biofilms by various microbes undergoes a number of phases till its maturation (Stoodley et al., 2002; Breyers and Ratner, 2004; Johnston, 2004). Thus, for accomplishment of the scenario it gradually passes through different steps, viz. 1. Attachment process (reversible and irreversible) 2. Biofilm polymer/microcolony formation 3. Maturation and replication and 4. Cell dispersion or detachment. Under these major steps, the following sequential steps are also involved in biofilm formation. These include- a) substratum pre-conditioning by ambient macromolecule; b) cell deposition; c) cell adsorption; d) desorption; e) cell-to-cell signalling and onset of exopolymer production; f) convective and diffusive transport of O2 and nutrients; g) replication and growth; h) secretion of polysaccharide matrix, and i) detachment, erosion and sloughing (Simoes et al., 2010).

The process begins with the adhesion of cells to the substratum and this adhesion to the adjoining surfaces is a crucial factor for the entire process (Vieira et al., 1993; Busscher et al., 1995; Donlan., 2002; Chae et al., 2006; Palmer et al., 2007; Patel et al., 2007; Oulahal et al., 2008;). The cell organelles like outer membrane proteins, capsular polysaccharide, lipopolysaccharides, curli, pilli, fimbrillae, prosthecae, stalks and flagella create impact on cell charge and hydrophobicity as well as guide the adhesion during biofilm formation (Morris et al., 1997; Harbron and Kent, 1988; Sauer and Camper, 2001; Daniels et al., 2004; De Rezende et al., 2005). Hence, this preliminary conjunction is reversible as the bacterial interaction is weak and the attachment entails numerous morphological changes which are essential for biofilm formation as a result, there lies a possibility of detachment of many cells (Terraf et al., 2012). Thereafter the attachment turns to irreversible one and the bonding is permanent due to exopolysaccharides (EPS) secretion and subsequently the microcolonies form and expand on the surface (Stoodley et al., 2002). These microcolonies form by the cell-aggregation which in turn occurs through growth of microbes, enabling EPS production (Chmielewski and Frank, 2006). Microbial cells remain entrenched within the EPS matrix in multiple layers (cohesion) which provide nutrition for them. A bacterial EPS encompasses polysaccharide, proteins, lipids, nucleic acid, phosphor lipids and humic substances and may also carry the water channels (Jahn and Nielsen et al., 1998). Thus, EPS proves to be a boon for microbes as it provides more resistance within a biofilm (Davies et al., 1998; Sauer and Camper, 2001; Parsek and Greenberg, 2005). Besides, the microcolony aids substrate exchange within species and removes their end-products (Costerton et al., 1994). Progressively maturation and ordered construction occurs in a time period and ultimately detachment of cells occurs and the colonization at new areas crop up (Sauer et al., 2002; Stoodley et al., 2002).

Adherence

Colonisation & Maturation

Release

Fig.1: Flow diagram showing the major steps of biofilm formation

Factors Affecting Biofilm Formation

Although the process of biofilm formation does not go so smooth every time. Sometimes it may influence by multiple factors like bacterial strains, pH, nutrient concentration or level, surface texture, surface hydrophobicity, temperature, speed of liquid stream, osmotic pressure etc which affect the biofilm creation (Donlan and Costerton, 2002; Donlan, 2002; Nilsson et al., 2011). The degree of biofilm formation also varies highly with the inter-strain variation, increase in temperature, flow velocity or nutrient concentration. Though these factors may impart negative effect on the formation process if their critical levels are being exceeded (Stoodley et al., 1999; Pan et al., 2009). A pictorial presentation of factors which affect to biofilm formation is given below:

Among the different bacterial genera the Bacillus is the predominant bacteria of the dairy plants as present in raw and even pasteurized milk because of its capability to produce heat-resistant spores (Wilkinson and Davies, 1973; Meer et al., 1991; Sharma and Anand, 2002; Ranieri et al., 2009; Shaheen et al., 2010). Within the Bacillus species, B. subtilis is the classical one which is able to form vigorous biofilms in dairy industry (Chu et al., 2006; Vlamakis et al., 2013). It requires mainly carbon and energy to make the biofilm and use a number of sugars, organic acids and different organic compounds for this task (Stanley et al., 2003; Chu et al., 2008; Fujita, 2009).

Pseudomonas

The genus Pseudomonas is another varied bacterial genera in which P. fluorescens is the most common one which is responsible for biofilm formation in the dairy processing units. It is well-known for this cause because of its high heat resistance and short generation time and these characteristics make it a successful biofilm former (Wiedmann et al., 2000; Pereira and Vieira, 2001; Dogan and Boor, 2003; Olofsson et al., 2007).

Listeria

Listeria monocytogenes is the chief player of the biofilm within this genera and its biofilm formation capability varies significantly among serotype/lineage and origin (di Bonaventura et al., 2008; Mu et al., 2014). Though the relationship between lineage and biofilm formation is controversial, as some of researcher states that strains of lineage I have more biofilm producing capacity than those of lineage II while som states just opposite to the above said (Djordjevic et al., 2002; Harvey et al., 2007; Combrouse et al., 2013). The biofilm creation by L. monocytogenes is mainly affected by temperature, strain origin and nutrient level (Nilsson et al., 2011). The L. monocytogenes also has the property of attachment to surfaces passively and its biofilms are primarily comprised of teichoic acids which can grow on polypropylene, steel, rubber and/or glass surfaces (Lemon et al., 2007; Silva et al., 2008; Tresse et al., 2009).

Staphylococcus

Staphylococcus is well recognised bacteria that may form biofilms on food contact surfaces in milk processing plants and especially Staphylococcus epidermidis has been recently described as a main biofilm forming species (Sharma & Anand, 2002; Vuong & Otto, 2002; Piette & Verschraegen, 2009). In the process of staphylococcal biofilm formation, the accumulation and development of a mature stage depend mainly on the polysaccharide intercellular adhesions (PIA) that promote bacterial accumulation, especially polysaccharide poly-N-succinylb- 1-6 glucosamine (PNAG) (Felipe et al., 2017).

Streptococcus

Mostly the cheese section of the dairy industry suffers the most due to the biofilms produced by Streptococcus, more specifically Streptococcus. thermophilus. In the heating chamber of the section where temperature remains within 30 to 730C lies, the maximum degree of biofilm formation occurs by the Streptococcus and thus pasteurized milk get contaminated (Couvigny et al., 2015). As a result the defects in milk and cheese quality like acidic flavour and undesirable texture are spotted (Hup et al., 1980; de Jong et al., 2002; Hood and Zottola, 1995; Palmer et al., 2007). Moreover, different strains of S. thermophilus from different dairy products show variable intensity of ability to produce biofilm ((Lortal et al., 2009; Couvigny et al., 2015; Scatassa et al., 2015).

Lactobacillus

Few strains of Lactobacillus like L. rhamnosus can form biofilms in vitro on the abiotic surfaces (glass or polystyrene) (Lebeer et al., 2007; Leccese et al., 2016). Unlike other bacteria, biofilm formation by Lactobacillus spp. is relatively beneficial because of its property of colonization and longer mucosal permanence of the host as these help in avoiding pathogenic bacterial colonization (Terraf et al., 2012).

coli

coli possesses the capacity to form biofilm structures both in vivo and in vitro. In fact among the facultative anaerobic bacteria of the GI tract, E. colican bloom in a multispecies biofilm environment having their structural characteristics (Costerton et al., 1995; Probert and Gibson, 2002). The autoinducer-2 (AI-2) of E. coli O157:H7 act as supplementary force for biofilm production as AI-2 signals regulate chemotaxis, flagellar synthesis and motility of genes (Pillai and Jesudhasan, 2006). The E. coli O157:H7 yields exopolysaccharides (EPS) which helps in cell attachment and formation of 3D structures of biofilms (Ozer and Demirci, 2006).

Miscellaneous

Besides the above one there are various other microbes which form the biofilms in dairy like obligate and facultative thermophiles. The Obligate thermophiles include Anoxybacillus, Flavithermus, Geobacillus spp. which prefer higher temperatures (40-680C) to grow and the facultative thermophiles B. lichenformis, B. coagulans, B. pumilus, B. sporothermodurans also involve in biofilm formation (Ronimus et al., 2003; Schelderman et al., 2005; Scott et al., 2007).

Public Health Significance of Biofilms

The biofilms contribute in the deterioration of food quality due to the action of various microbial enzymes like lipases, proteases. In the dairy sector proteases enzyme play the major role in this regard and it is produced enormously by the different genera of bacteria (Celestino et al., 1997; Santos et al., 2003). Because of the catalytic reactions the biofilms persuade corrosion of metallic food surfaces (Vieira et al., 1993). Also, the microbial quality of the products becomes inferior due to remarkable decrease in the heat transfer efficiency of the surfaces (Mittelman, 1998). The bacterial biofilms can persist in dairy plants and can potentially reduce the self-life of the pasteurized milk, cream, cheese and the other milk products (Gopal et al., 2015; López et al., 2015; Tschiedel et al., 2015). Thus, majority of the food borne bacterial contamination leads to gastroenteritis creating a serious threat to public health and disbalance to the livelihood of mankind.

Prevention and Control Strategy of Biofilms

The easiest, cheapest and the most common measure of arresting the biofilm in the dairy industry is cleaning and disinfecting of the all sites, equipment and instruments especially in the alarming zones (Simões et al., 2006). It prevent the development and spread of biofilms on the surfaces and in the food items (Gibson et al., 1999; Verran, 2002). The efficiency and intensity of cleaning directly affect the final quality of the prepared products (Bremer et al., 2006). The poor cleaning procedures are the causes of retention of biofilms as disinfectants are unable to enter the matrix of biofilms (Simões et al., 2006).

In recent times, many natural compounds like various plant extracts, honey, essential oil (EO) etc. are found effective against microbial biofilm formation. These different natural products are used efficiently against different microbial biofilms in various successful experiments. For example, honey is a natural product having antimicrobial properties against about 60 species of bacteria and fungi (Massocks et al., 2012; Santangelo, 2013; Molan, 2013). It was found that honey helped in inhibiting Enterococcus spp. biofilm production and reducing biofilm formation of EHEC O157:H7 (Lee, 2011; Ng et al., 2014). Even a low concentration of honey can arrest the curling QS expression and virulence genes in bacteria (Lee, 2011). Similarly, essential oils (EOs) are also used against a wide range of pathogens since time immemorial as no antimicrobial resistance occurs (Hammer et al., 1999; Ohno et al., 2003; Ali et al., 2005). But now these are recognised too as an antibiofilm agent (Isman, 2000). As cumin oil and cinnamon oil, both are used in food industry for their aroma, are now also a potent antibiofilmers (Chang et al., 2001; Iacobellis et al., 2005). A few of such natural products are listed in the below table.

Besides the plant extract, bio-cleaners (enzyme-based detergents) or green chemicals help extensively in this field and practice of mixture of enzymes promote quicker degradation of biofilm (Sim˜oes et al., 2010). Likewise, phages having polysaccharide degrading enzymes can destroy biofilms rapidly and up to 80% of Pseudomonas fluorescens biofilms can be destroyed by phages and the bacteriophage T4 found to be effective against E. coli biofilms (Doolittle et al., 1995; Sillankorva et al., 2004). Biosurfactants also use as preventive measures as biosurfactants from Lactococcus lactis 53 inhibit biofilm formation on silicon rubber of the equipments (Rodrigues et al., 2004). The Surfactin produced by Bacillus subtilis prevents Salmonella enterica, E. coli and Proteus mirabilis biofilms (Mireles et al., 2001). In dairy processing sectors biopreservatives like nisin, lauricidin, reuterin, pediocin protect from biofilm formation by various microbes including L. monocytogenes (Dufour et al., 2004; Zhao et al., 2004). Moreover, quorum sensing property of the microbes can be inhibited in order to prevent biofilm production at its very early stage (Dong et al., 2002). The use of ultrasound is one of the most recent advances to control and prevent microbial biofilm generation (Kallioinen and Manttari, 2011).

Conclusion

Biofilm formation possesses profound implications and throws a major challenge to the dairy sector where they act as the principal reservoir of microbial contamination. These lead to financial crisis by impairment of raw material and its products. Therefore, choosing of a profound, prominent and efficient measure is in an urge in order to safeguard the whole sector from further deficiency and mitigating the present problem judiciously. The decision adopted should be in harmony with science, capital and time so that no turbulence can slow down its way.

De Jong, P., Te Giffel, M.C. and Kiezebrink, E.A. (2002). Prediction of the adherence, growth and release of microorganisms in production chains. International Journal of Food Microbiology.74:13–25. doi:10.1016/S0168-1605(01)00713-9.

Dufour, M., Simmonds, R.S. and Bremer, P.J. (2004). Development of a laboratory scale clean-in-place system to test the effectiveness of “natural” antimicrobials against dairy biofilms. Journal of Food Protection, 67, 1438-1443.

Durango, J., Arrieta, G. and Mattar, S. (2004). Presence of Salmonella as a risk to public health in the Caribbean zone of Colombia. Biomedica, 24, 89–96.

Oliveira, M.M., Brugnera, D.F., Nasciment, J.D., Batista, N.N. and Piccoli, R.H. (2012). Cinnamon essential oil and cinamaldehyde in the control of bacterial bioﬁlm formed on stainless steel surfaces. European Food Research and Technology, 234,821–32.

Pereira, M.O. and Vieira, M.J. (2001). Effects of the interactions between glutaraldehyde and the polymeric matrix on the efficacy of the biocide against Pseudomonas fluorescens biofilms. Biofouling, 17, 93–101.

Tresse, O., Lebret, V., Garmyn, D. and Dussurget, O. (2009). The impact of growth history and flagellation on the adhesion of various Listeria monocytogenes strains to polystyrene. Canadian Journal of Microbiology. 55: 189–196.